Self-gelling alginate systems and uses thereof

ABSTRACT

Kits and compositions for producing an alginate gel are disclosed. The kits and compositions comprise soluble alginate and insoluble alginate/gelling ion particles. Methods for dispensing a self-gelling alginate dispersion are disclosed. The methods comprise forming a dispersion of insoluble alginate/gelling ion particles in a solution containing soluble alginate, and dispensing the dispersion whereby the dispersion forms an alginate gel matrix. The methods may include dispensing the dispersion into the body of an individual. An alginate gel having a thickness of greater than 5 mm and a homogenous alginate matrix network and homogenous alginate gels free of one or more of: sulfates citrates, phosphates, lactatates, EDTA or lipids are disclosed. Implantable devices comprising a homogenous alginate gel coating are disclosed. Methods of improving the viability of pancreatic islets, or other cellular aggregates or tissue, following isolation and during storage and transport are disclosed.

FIELD OF THE INVENTION

The present invention relates to alginate systems which have a delayedgelling process and to compositions, devices, kits and methods of makingand using such systems.

BACKGROUND OF THE INVENTION

This application claims priority to U.S. Provisional Application Ser.No. 60/617,852 entitled Self-Gelling Alginate Systems and Uses Thereof,filed Oct. 12, 2004, which is incorporated herein by reference.

Alginates are hydrophilic marine biopolymers with the unique ability toform heat-stable gels that can develop and set at physiologicallyrelevant temperatures. Alginates are a family of non-branched binarycopolymers of 1-4 glycosidically linked β-D-mannuronic acid (M) andα-L-guluronic acid (G) residues. The relative amount of the two uronicacid monomers and their sequential arrangement along the polymer chainvary widely, depending on the origin of the alginate. Alginate is thestructural polymer in marine brown algae such as Laminaria hyperborea,Macrocystis pyrifera, Lessonia nigrescens and Ascophyllum nodosum.Alginate is also produced by certain bacteria such as Pseudomonasaeruginosa, Azotobacter vinelandii and Pseudomonas fluorescens(WO04011628 A1).

Alginate gels are produced when a divalent cation forms ionic bonds withthe negatively charged group from a G residue from each of two differentalginate polymers, thereby cross-linking the two polymers. The formationof multiple cross-linkages among numerous alginate polymers results inthe matrix that is the alginate gel structure.

Alginate gels can be hydrogels, i.e. cross-linked alginate polymers thatcontain large amounts of water without dissolution. Biopolymer gels,such as alginate hydrogels are attractive candidates for tissueengineering and other biomedical applications. Because of this and theability to form gels under physiologic conditions, alginates are widelyused and studied for encapsulation purposes and as a biostructurematerial. The entrapment of cells in alginate beads is a commonly usedtechnique. Also alginates have been shown to be useful material forother types of biostructures, including tissue engineering applicationsand as scaffolds for nerve regenerations.

Different methods for making alginate hydrogels exist. The most commonmethod is the dialysis/diffusion method where the alginate solution isgelled by diffusion of gelling ions from an outer reservoir. This methodis mostly used when making alginate gel beads and in food applications.The manufacturing of alginate microbeads is a rapid process limited bythe diffusion of gelling ions into the gel network. Although thisprocess is well suitable for entrapment of cells in microbeads, it isless useful for the production of other shapes or structures. Formanufacturing of gel structures of larger size diffusion gelling systemsmay have limited possibility. This is because the rapid gelling processlimits the time to allow shaping of the gel structure.

A delay in the gelling process may be used to allow for the injection ofsolutions into the body and/or to mix cells or other biomaterial intothe gel matrix prior to the gel forming. Therefore, alternative methodshave been developed for the manufacturing of other types ofbiocompatible alginate gel structures. The gelling speed may be reducedby using internal gelling systems of which the gelling ions are releasedmore slowly inside the forming gel. This is described as internalsetting of the gel. Commonly, in an internal gelling system, a calciumsalt with limited solubility, or complexed Ca²⁺ ions, are mixed with analginate solution into which the calcium ions are slowly released.Calcium sulfate has been used in alginate based cell delivery vehiclesfor tissue engineering. The release of calcium and gelling kinetics mayalso be controlled by using calcium salts with pH dependent solubilityand the addition of a slowly acting acid such as D-glucono-δ-lactone(GDL). As the pH changes, calcium ions are released. Also calciumcontaining liposomes have been used as a controllable alginate gellingsystem. Alginate gel systems based upon internal gelling may have a moredefined and limited supply of gelling ions as opposed to diffusionsystems where calcium ions are allowed to diffuse into the alginatesolution to give a calcium saturated gel.

Current methods for manufacturing of alginate gel structures havelimitations. Some techniques are only useful to make gels of limitedsizes and shapes. Depending of the applications there may problemsassociated with the control of the gelling kinetics. In some case,undesirable materials are present in gels because such materials areresidues and by-products of chemically controlled gelling mechanisms. Insome cases, non-physiologic pH values are required for gelling and suchconditions may present limitations to the use of such methods. There istherefore a need for other gelling systems and formulations.

SUMMARY OF THE INVENTION

The present invention relates to kits for producing an alginate gel. Thekits comprise a first container comprising soluble alginate, and asecond container comprising insoluble alginate/gelling ion particles.

The present invention further relates to compositions for preparingalginate gels. The compositions comprise immediately soluble alginateand insoluble alginate/gelling ion particles.

The present invention further relates to methods for dispensingself-gelling alginate dispersion. The methods comprise forming adispersion of insoluble alginate/gelling ion particles in a solution ofsoluble alginate, and dispensing the dispersion whereby the dispersionforms an alginate gel matrix.

The present invention further relates to methods for dispensingself-gelling alginate dispersion into an individual. The methodscomprise forming a dispersion of insoluble alginate/gelling ionparticles in a solution of soluble alginate, and dispensing thedispersion into an individual whereby the dispersion forms an alginategel matrix in the individual.

The present invention further relates to methods for dispensingself-gelling alginate dispersion into an individual for use as tissuebulking material, for use in a vascular embolization procedure, for useto prevent post surgical adhesion formation, for use in wound treatmentprocedures, for use in diabetes treatments and for use in treatment ofarthritis.

The present invention further relates to methods of using an implantablealginate gel. The methods comprise forming self gelling alginate bydispensing self gelling alginate dispersion and, following gelformation, implanting the implantable alginate gel into an individual.

The present invention further relates to methods of producingimplantable devices.

The present invention further relates to alginate gels having athickness of greater than 5 mm and a homogenous alginate matrix network.

The present invention further relates to alginate gels having athickness of greater than 5 mm and free of one or more of: sulfatescitrates, phosphates, lactatates, EDTA or lipids.

The present invention further relates to implantable devices comprisinga homogenous alginate gel coating.

The present invention further relates to methods filling or repairingosteochondral defects resulting from osteoarthritis by dispensing a selfgelling alginate dispersion that includes chondrocytes into anindividual's body or by implanting a biocompatible matrix that includeschondrocytes into an individual's body

The present invention further relates to methods of treating diabetes bydispensing a self gelling alginate dispersion that includesinsulin-producing cells or multicellular aggregates into an individual'sbody or by implanting a biocompatible matrix that includesinsulin-producing cells or multicellular aggregates into an individual'sbody.

The present invention further relates to methods of improving theviability of pancreatic islets, or other cellular aggregates or tissue,following isolation and during storage and transport by incorporatingthe islets, or cellular aggregates or tissue into a self gellingalginate dispersion.

The present invention further relates to ultrapure insolublealginate/gelling ion particles and methods of producing the same.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 contains data from rheometer measurements. Oscillationmeasurements were performed using a Physica MCR300 rheometer. Storagemodulus as a function of time for gels with the following differentcalcium-alginate (Protaweld TX 120) concentrations are shown(concentrations in the mixture/gel): 1.0% calcium-alginate mixed with1.0% sodium-alginate, and 1.5% calcium-alginate mixed with 1.0%sodium-alginate, and 2.0% calcium-alginate mixed with 1.0%sodium-alginate. The sodium alginate used was Protanal SF 120.

FIG. 2 contains data from rheometer measurements. Oscillationmeasurements were performed using a Physica MCR300 rheometer. Storagemodulus as a function of time for gels made by mixing equal amounts ofsodium alginate (Protanal SF 120) and calcium alginate (Protaweld TX120) at the following concentrations in the gel: 0.75%, 1%, 1.25% or1.5% sodium alginate and calcium alginate

FIG. 3 contains data from rheometer measurements. Oscillationmeasurements were performed using a Physica MCR300 rheometer. Storagemodulus was measured as a function of time for gels containing differentmolecular weights of calcium alginates (panel A) and sodium alginates(panel B) are shown as follows. The gels in panel A contain 1% sodiumalginate and 1.5% calcium alginate and the gels in panel B contain 1%calcium alginate and 1% sodium alginate. The alginates used in panel Awere: Calcium alginate: Protaweld TX 120 and Protaweld TX 120 degradedfor 33 days at 60°. The viscosity (1% solution at 20° C.) of the twoCa-alginates, measured as sodium alginate, was 270 mPas and 44 mPas,respectively. Sodium alginate: Protanal SF 120. The alginates used inpanel B were: Calcium alginate: Protaweld TX 120. Sodium alginate:Protanal SF 120 and Protanal SF/LF. The viscosity (1% solution at 20°C.) of the sodium alginates was 95 mPas and 355 mPas, respectively.

FIG. 4 contains data from rheometer measurements. Oscillationmeasurements were performed using a Physica MCR300 rheometer. Storagemodulus as a function of time for gels made with strontium or calcium asgelling ions. The amount of sodium alginate (Protanal SF 120) andstrontium/calcium alginate was each adjusted to be 0.75% in the gel. Thecalcium alginate used was made by kneading alginic acid (FMC processproduct) (65.2 g) with calcium carbonate (35.32 g) in a lab kneader for1 hour, then drying and milling. The strontium alginate used was made bykneading alginic acid (FMC process product) (65.2 g) with strontiumcarbonate (52.10 g) in a lab kneader for 1.5 hour, then drying andmilling.

FIG. 5 contains data from rheometer measurements. Oscillationmeasurements were performed using a Physica MCR300 rheometer. Storagemodulus as a function of time for gels containing sodium alginate withhigh and low content of guluronic acid. In FIG. 5, panel A, calciumalginate (Protaweld TX 120) and sodium alginate used was each adjustedto be 1.0% of the gel. The sodium alginates used were Protanal SF 120(69% G) and Protanal HF 60D (32% G, MW: 119 000). In FIG. 5, panel B,5.5% strontium alginate (Example 14) was mixed with 1.25% sodiumalginate at a ratio of 1:4 (final alginate concentration was 2.1%). Thesodium alginates used were PRONOVA UP 100G (69% G, MW: 122 000) andPRONOVA UP 100M (46% G, MW: 119 000). The MW (and viscosity) of the twosodium alginate batches was selected to be similar (as close aspossible). Each curve in FIG. 5, panel B, is the mean of threeindependent measurements (curves) with standard error of the mean shownfor each point.

FIG. 6 shows stability and biodegradability for alginate gels made withdifferent content of calcium ions and stored for 6 months underphysiologic conditions. Gels discs were made by mixing an autoclavedcalcium alginate dispersion (Protaweld TX 120) and sterile filteredsodium alginate (PRONOVA UP LVG) to a final concentration of 1.0%alginate each and the dispersion was gelled in the two Petri dishes. Thegel discs in one dish (marked V) was washed with 50 mM calcium chloridefor 10 minutes after gelling and both dishes was thereafter added cellculture medium (DMEM supplemented with 10% FBS). The dishes were storedunder sterile conditions in a CO₂-incubator and the medium was changedregularly three times a week. The size of the largest gel disc in eachdish was initially of the same size. The picture shown was taken aftersix months.

FIG. 7 shows data from experiments using cells entrapped in alginateself-gelling alginate. FIG. 7, panel A, shows C2C12 mouse myoblast cells45 days after entrapment in self-gelling alginate gel. The gel was madeand stored similar as in FIG. 6 but included the cells. The picture wastaken using a fluorescence microscope after staining the cells withcalcein (Molecular Probes, L-3224) as a marker of cell viability.Enlightened areas and spots shows the presence of viable cells. Viablecells are located inside and on the surface of the gel construct. FIG.7, panel B, shows human chondrocytes entrapped in alginate self-gel. Thegel was made of 5 ml mixed self-gel of PRONOVA SLG 20 and calciumalginate (Example 14) containing human chondrocytes. Three days aftergelling the gels were sectioned into 600 μm slices using a vibratome.The gel slices was stored in cell growth medium in a CO₂-incubator andthe picture was taken after six months. The picture was taken using afluorescence microscope after staining the cells with calcein (MolecularProbes, L-3224) for viability and clearly shows the presence of a highnumber of viable cells (enlightened spots).

FIG. 8 contains data from rheometer measurements. Oscillationmeasurements were performed using a Physica MCR300 rheometer. Storagemodulus as a function of time for gels containing 1.25% sodium alginate(PRONOVA UP 100 G) mixed with 5.5% strontium alginate (Example 14) at aratio of 4:1 (final alginate concentration was 2.1%) in the presence orabsence of sodium chloride or sodium hexametaphosphate. Each curve isthe mean of three independent measurements (curves) with standard errorof the mean shown for each point.

FIG. 9 contains data from rheometer measurements. Oscillationmeasurements were performed using a Physica MCR300 rheometer. Storagemodulus as a function of time for gels containing 1.25% sodium alginate(PRONOVA UP 100 G) mixed with 5.5% calcium alginate (Example 14)manufactured at different particle sizes at a ratio of 4:1 (final totalalginate concentration was 2.1%). The different particles sizes weremade by milling freeze dried calcium alginate and sifting at the sizesindicated. Each curve is the mean of three independent measurements withstandard error of the mean shown for each point.

FIG. 10 contains data from rheometer measurements. Oscillationmeasurements were performed using a Physica MCR300 rheometer. Storagemodulus as a function of time for gels containing 1.25% sodium alginate(PRONOVA UP LVG) mixed with 10% calcium alginate (Example 14) atdifferent temperatures at a ratio of 9:1 (final alginate concentrationwas 2%).

FIG. 11 contains data from rheometer measurements. Oscillationmeasurements were performed using a Physica MCR300 rheometer. Storagemodulus was measured as a function of time for gels containing differentmolecular weights of sodium alginate. The gels contain 1.25% sodiumalginate (PRONOVA UP100G) not degraded (control) or the same alginatebatch degraded. A gel was also made of the degraded sodium alginate at aconcentration of 2.5% (upper curve). In all cases the sodium alginateswere mixed with 5.5% strontium alginate (Example 14) at a ratio of 4:1.Each curve is the mean of three independent measurements (curves) withstandard error of the mean shown for each point.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

An alternative alginate gelling system is provided. This system is usedin numerous biomedical applications as well as other applications. Thesystem can include alginate and gelling ions that have a high degree ofbiocompatibility. The system provides for variations in gelling time andgel strength depending upon the needs of a specific application. Thesystem provides gelling without the pH changes associated with othersystems and requires a minimum number of components.

The system comprises two components: one is soluble alginate; the otheris insoluble alginate/gelling ion particles. When the components arecombined in the presence of a solvent to form a dispersion, an alginategel begins to form as the gelling ion of the particles begins crosslinking alginate polymers from the particles and the soluble alginatepolymers in solution. The two components may be mixed by stirring or byusing a suitable mixing device. The gelling kinetics of the formulationare dependent upon several factors including: the concentration of thesoluble alginate in solution, the concentration of the insolublealginate particles in the dispersion, the relative content of gellingion to alginate, the presence of non-gelling ions or othercarbohydrates, temperature, the size of insoluble alginate/gelling ionparticles, the content of cells, multicellular aggregates, tissues orother biomaterials to be entrapped in the gel or present during gelling,(the presence of impurities) and the types of alginates used, as well asthe manufacturing process for the insoluble alginate particles and postmanufacturing treatment of alginate starting materials. This alginatesystem may therefore be widely adapted to each particular application.For entrapment of cells, multicellular aggregates, tissues or otherbiomaterials within the forming gel, the solvent, the alginate solutionor dispersion may be premixed with the material to be entrapped.

The dispersion may be dispensed within an individual as a liquid/slurryto a site where an alginate gel matrix is desirable. Alginate gelformation, initiated when the soluble alginate and insolublealginate/gelling ion particles are mixed in the presence of a solvent,continues and the alginate gel sets in situ. As used herein, the term“self-gelling” is meant to refer to the gelling process which occurswhen the soluble alginate and insoluble alginate/gelling ion particlesare mixed in the presence of a solvent. A “self gelling alginate” is analginate dispersion or gel which includes or is formed by solublealginate and insoluble alginate/gelling ion particles in a solvent.

The components used in the system may be maintained prior to use in anyof several forms. For example, the soluble alginate may be maintained insolution or as a powder. In some embodiments, the soluble alginate maybe maintained as a powder that is immediately soluble such as when it isfreeze dried. Similarly, the insoluble alginate/gelling ion particlesmay be maintained as a dispersion or as a powder.

The alginate polymers or combinations thereof used in the solublealginate may be the same or different from those in the insolublealginate/gelling ion particles.

The concentration of alginate, both soluble alginate and the alginate inthe form of insoluble alginate/gelling ion particles in a dispersionrelative to the amount of solvent affects gelling time, porosity,stability and biodegradability, gel strength and elasticity of gels andgels having specific properties may be prepared by using specific ratiosof soluble alginate and insoluble alginate/gelling ion particles tosolvent. Generally, the lower the concentration of alginate (for a givenratio of soluble alginate to insoluble alginate), the more biodegradablea gel will be. In some embodiments, approximately 0.5%, 0.75%, 1%,1.25%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more alginate(soluble alginate and alginate in the form of insoluble alginate/gellingion particles) may be used.

The relative concentration of the soluble alginate to alginate in theform of insoluble alginate/gelling ion particles in the dispersionaffects gelling time, pore size, gel strength and elasticity of gels aswell as stability and biodegradability and gels having specificproperties may be prepared by using specific ratios of soluble alginateto insoluble alginate/gelling ion particles. In some embodiments, theconcentration of soluble alginate is approximately equal toconcentration of alginate in the form of insoluble alginate/gelling ionparticles (1:1). In some embodiments, the concentration of alginate inthe form of insoluble alginate/gelling ion particles is twice that ofsoluble alginate (2:1). In some embodiments, the concentration ofalginate in the form of insoluble alginate/gelling ion particles is halfthat of soluble alginate (1:2). In some embodiments, the concentrationof alginate in the form of insoluble alginate/gelling ion particles tosoluble alginate is 1 to 0.7 (1:0.7). Generally, the less gelling ionpresent, the more biodegradable a gel will be. Reducing theconcentration of insoluble alginate/gelling ion in the system may beused to create gels with lower stability and higher biodegradability asthe gel network is less saturated with cross-linking ions. Self gellingallows for the preparation of gels with lower concentrations of gellingion to produce gels particularly well suited for biodegradable uses. Insome preferred embodiments, alginate ratios of insolublealginate/gelling ion particles to soluble alginate are: 5:1, 4:1, 3:1,2:1, 1:1, 1:2, 1:3, 1:4 or 1:5.

The relative content of G and M monomers in the alginate polymersaffects pore size, stability and biodegradability, gel strength andelasticity of gels. Alginate polymers contains large variations in thetotal content of M and G, and the relative content of sequencestructures also varies largely (G-blocks, M-blocks and MG alternatingsequences) as well as the length of the sequences along the polymerchain. Generally, the lower the G content relative to M content in thealginate polymers used the more biodegradable a gel will be. Gels withhigh G content alginate generally have larger pore sizes and strongergel strength relative to gels with high M alginate, which have smallerpore sizes and lower gel strength. In some embodiments, one or more ofthe alginate polymers of the alginate matrix contain more than 50%α-L-guluronic acid. In some embodiments, one or more of the alginatepolymers of the alginate matrix contain more than 60% α-L-guluronicacid. In some embodiments, one or more of the alginate polymers of thealginate matrix contain 60% to 80% α-L-guluronic acid. In someembodiments, one or more of the alginate polymers of the alginate matrixcontain 65% to 75% α-L-guluronic acid. In some embodiments, one or moreof the alginate polymers of the alginate matrix contain more than 70%α-L-guluronic acid. In some embodiments, one or more of the alginatepolymers of the alginate matrix contain more than 50% C-5 epimerβ-D-mannuronic acid. In some embodiments, one or more of the alginatepolymers of the alginate matrix contain more than 60% C-5 epimerβ-D-mannuronic acid. In some embodiments, one or more of the alginatepolymers of the alginate matrix contain 60% to 80% C-5 epimerβ-D-mannuronic acid. In some embodiments, one or more of the alginatepolymers of the alginate matrix contain 65% to 75% C-5 epimerβ-D-mannuronic acid. In some embodiments, one or more of the alginatepolymers of the alginate matrix contain more than 70% C-5 epimerβ-D-mannuronic acid. Procedures for producing uronic blocks from aredisclosed in U.S. Pat. No. 6,121,441. G-block alginate polymers andtheir uses as modulators of alginate gel properties is set forth in U.S.Pat. No. 6,407,226. Some preferred embodiments, 30% G, 35% G, 40% G, 45%G, 50% G, 55% G, 60% G, 65% G, 70% G, 75%, 80% G or 85% G.

The average molecular weight of alginate polymers affects gelling time,pore size, gel strength and elasticity of gels. Alginate polymers mayhave average molecular weights ranging from 2 to 1000 kD. The molecularweight of alginates may affect gel formation and the final gelproperties. Generally, the lower the molecular weight of the alginateused the more biodegradable a gel will be. The alginate polymers orcombinations thereof used in the soluble alginate components may be thesame or different from the polymers or combinations thereof used in theinsoluble alginate/gelling ion particles. In some embodiments, thealginate polymers of the alginate matrix have an average molecule weightof from 5 to 350 kD. In some embodiments, the alginate polymers of thealginate matrix have an average molecule weight of from 2 to 100 kD. Insome embodiments, the alginate polymers of the alginate matrix have anaverage molecule weight of from 50 to 500 kD. In some embodiments, thealginate polymers of the alginate matrix have an average molecule weightof from 100 to 1000 kD. In some embodiments, gels are designed to have ahigh degree of biodegradability. Accordingly, gels having less alginate,less gelling ion lower G content and lower molecular weight alginatescan be produced using the lower limits of one or more of theseparameters as set forth herein to produce gels with a high degree ofbiodegradability.

The alginate may possess a viscosity in a 1% solution measured at 20degrees centigrade of from 25 to 1000 mPas and in some embodiments,preferentially 50 to 1000 mPas (1% solution, 20 C.).

In some embodiments, it is preferred that methods of manufacture ofinsoluble alginate/gelling ion particles provide products withstoichiometric amount (100% saturation) of gelling ion. Use of suchstoichiometric salts imparts greater reproducibility in the self-gellingalginate systems. In some embodiments, it is preferred that method ofmanufacture of insoluble alginate/gelling ion particles provide productswith sub-stoichiometric amount (<100% saturation) of said gelling ion.Use of such sub-stoichiometric salts imparts biodegradability toself-gelling alginate systems.

In some embodiments, the alginate is ultrapure alginate. Ultrapurealginate is commercially available such as from different sources ofseaweed like Laminaria Hyperborea. Commercial calcium salts of alginicacid are generally manufactured in processes whereby calcium is added toalginic acid in the solid phase by simple admixture and kneading of thecomponents together. Examples of commercially available calcium salts ofalginic acid are Protaweld (from FMC BioPolymer) and Kelset from ISPCorporation. The insoluble alginate/gelling ion particles may beproduced using ultrapure alginate by making an alginate gel using theultrapure alginate and a gelling ion, washing out sodium or other ionsthat were present in the ultrapure alginate, drying the gel to removethe water, and making particles from the dried gel. In some embodiments,the insoluble alginate/gelling ion particles are stoichiometric salts.Insoluble alginate/gelling ion particles preferably have a high purityand a specific, consistent and generally uniform content of gelling ionsuch as, for example, calcium or strontium barium, zinc, iron,manganese, copper, lead, cobalt, nickel, or combinations thereof, suchthat gel formation speed and gel strength can be provided with moreprecise predictability. Insoluble alkaline earth salts of alginic acidsuch as for example calcium alginate or strontium alginate (dependingupon the gelling ion used) or insoluble transition metal salts ofalginic acid (such as those using gelling ions of copper, nickel, zinc,lead, iron, manganese or cobalt) can be manufactured with a known andpredetermined content of alkaline earth ions by precipitation from thesolutions. In some embodiments, commercially available sodium alginateis first used to prepare a sodium alginate solution. Optionally, sodiumsalt such as sodium carbonate may be included in the sodium alginatesolution. A salt containing the desired gelling ion for the insolublealginate/gelling ion particle, such as for example, calcium salt orstrontium salt such as calcium chloride or strontium chloride, is usedto make a solution. The sodium alginate solution is combined, preferablyslowly, with the gelling ion solution. Preferably, the combinedsolutions are continuously stirred during the mixing process. Insolublealginate such as for example calcium alginate or strontium alginate(depending upon the gelling ion used) precipitates from the combinedsolutions. The precipitated insoluble alginate is then be removed fromthe solution and washed repeatedly, such as 2-10 times, with purifiedwater for example to remove all soluble ions. The removal of solubleions is confirmed for example by testing the conductivity of insolublealginate in purified water compared to the conductivity of purifiedwater. After washing, the insoluble alginate can be dried, such as witha vacuum. The dried alginate can be milled and, in some embodiments,selected for particle sizes.

In some embodiments, the alginate is sterile. In some preferredembodiments, the alginate is sterile ultrapure alginate. Conditionsoften used to sterilize material can change the alginate, such asdecrease the molecular weight. In some embodiments, the sterile alginateis produced using sterility filters. In some embodiments, the alignatehas an endotoxin level of <25 EU/gram.

In some embodiments, the alginate matrix is may be coated with apolycationic polymer like a poly amino acid or chitosan after the gelmatrix forms. In some embodiments, poly-lysine is the polycationicpolymer. In some embodiments, poly-lysine is linked to another moietyand the poly-lysine is thus used to facilitate association of the moietyto the gel. Examples of moieties linked to the gel using polycationicpolymers include, for example, drugs, peptides, contrast reagents,receptor binding ligands or other detectable labels. Some specificexamples include vascular endothelial growth factor (VEGF), epidermalgrowth factor (EGF), transforming growth factor (TGF), and bonemorphogenic protein (BMP). Drugs may include cancer chemotherapeuticagents such as Taxol, cis-platin and/or other platinum-containingderivatives. Carbohydrate polymers may include hyaluronan, chitosan,heparin, laminarin, fucoidan, chondroitin sulfate. In some embodiments,the alginates used are modified alginate polymers such as chemicallymodified alginate in which one or more polymers are linked to adifferent alginate polymer. Examples of such modified alginate polymersmay be found in U.S. Pat. No. 6,642,363, which is incorporated herein byreference.

In some embodiments, the alginate polymer may include a non-alginatemoiety such as, for example, a drug, a peptide, a contrast reagent, areceptor binding ligand or other detectable label. In one embodiment,the alginate polymer includes an RDG peptide (Arg-Asp-Gly), aradioactive moiety (e.g. ¹³¹I) or a radio opaque substance. Otherexamples of moieties linked to alginate polymers include, for example,drugs, peptides, contrast reagents, receptor binding ligands or otherdetectable labels. Some specific examples include vascular endothelialgrowth factor (VEGF), epidermal growth factor (EGF), transforming growthfactor (TGF), and bone morphogenic protein (BMP). Drugs may includecancer chemotherapeutic agents such as Taxol, cis-platin and/or otherplatinum-containing derivatives. Carbohydrate polymers may includehyaluronan, chitosan, heparin, laminarin, fucoidan, chondroitin sulfate.

The soluble alginate may be a salt such as, for example, Na⁺-alginate,K⁺-alginate, PEG-alginate (polyethylene glycol-alginate), NH₄-alginateor combinations thereof.

In some embodiments, the soluble alginate is freeze dried or otherwisedesiccated. Freeze dried soluble alginate is “immediately soluble.”“Immediately soluble” alginate is soluble in water in less than oneminute, preferably less than 30 seconds, more preferably less than 15seconds. “Readily soluble” alginate takes more than one minute andusually several minutes to go into solution.

The gelling ions used in the insoluble alginate/gelling ion particlesaffects gelling kinetics, gel strength, and elasticity. Gelling ionsalso have affects on cell growth. The gelling ions used in the insolublealginate/gelling ion particles may be Ca⁺⁺, Sr⁺⁺, Ba⁺⁺, Zn⁺⁺, Fe⁺⁺,Mn⁺⁺, Cu⁺⁺, Pb, Co, Ni, or combinations thereof.

The insoluble alginate gelling ion complexes are particles. Theparticles are generally non fibrous based on a L/D ratio where theparticle shape is characterized by a largest dimension (L) and smallestdimension (D). Non-fibrous L/D is less than 10, preferably less than 5,preferably less than 2. An L/D of 10 or more is a chopped fiber. Theinsoluble alginate gelling ion can be maintained as a dispersion or indry form. If the former, the dispersion can be mixed with a solutioncontaining soluble alginate or with immediately soluble alginate to forma dispersion of insoluble alginate/gelling ion particles in a solutioncontaining soluble alginate. If the insoluble alginate gelling ionparticles are in dry form, they may be mixed with dry immediatelysoluble alginate and subsequently with a solution to form a dispersionof insoluble alginate/gelling ion particles in a solution containingsoluble alginate or the dry insoluble alginate gelling ion particles maybe combined with a solution containing soluble alginate to form adispersion of insoluble alginate gelling ion particles in a solutioncontaining soluble alginate.

The agitation that occurs upon mixing the components to form thedispersion results in distribution of the solid particles within thesolution. The dispersion so produced can be in the form of a slurrywhich can be poured, injected and otherwise self gel within a mold orcavity to form the shape of such mold or cavity.

The dispersion of insoluble alginate gelling ion particles in a solutioncontaining soluble alginate is formed, it is dispensed to the site wherethe self gelling occurs to form an alginate gel. In some embodiments,the dispersion is dispensed to a site in vivo. In some embodiments, thedispersion is dispensed on to a site on an individual's body. In someembodiments, the dispersion is dispensed into a mold or other containeror surface.

The concentration of gelling ions used in the insoluble alginate/gellingion particles affects gelling kinetics, gel strength, and elasticity.The higher the concentration of gelling ions, the higher the gelstrength. Gel strength is highest when the gel is saturated with gellingion. Conversely, the lower the concentration of gelling ion, the lowerthe gel strength and higher the degree of biodegradability.

The particle size of the insoluble alginate/gelling ion particles mayaffects gelling kinetics and the final properties of the gel. Thesmaller the particle size the more rapid the completion of gelformation. Larger particle sizes produce stronger gels. Particle sizesmay be controlled by, for example, sifting insoluble alginate/gellingion particles through various different size filters such that theparticles can be generally all be within a predetermined size range. Insome embodiments, particles are <25 μm, 25-45 μm, 45-75 μm, 75-125 μmor >125 μm.

The solvent used may be, for example, water, saline, sugar solution,cell culture solution, a solution such as a drug solution, protein, ornucleic acid solution, a suspension such as a cell suspension,liposomes, or a contrast reagent suspension.

The alginate hydrogel formed may comprise, for example, drugs nucleicacid molecules, cells, multicellular aggregates, tissue, proteins,enzymes, liposomes, a contrast reagent or a biologically activematerial. Examples of a biologically active material are hyaluronate andchitosan. Contrast reagents include tantalum and gadolinium. Somespecific examples of proteins include vascular endothelial growth factor(VEGF), epidermal growth factor (EGF), transforming growth factor (TGF),and bone morphogenic protein (BMP). Drugs may include cancerchemotherapeutic agents such as Taxol, cis-platin and/or otherplatinum-containing derivatives. Carbohydrate polymers may includehyaluronan, chitosan, heparin, laminarin, fucoidan, chondroitin sulfate.

The cells that can be used in the gels include non-recombinant andrecombinant cells. In some embodiments in which cells are encapsulatedwithin an alginate matrix, encapsulated cells are mammalian cells,preferably human cells. In some embodiments in which encapsulated cellsare non-proliferating cells, the non-proliferating cells may be selectedfrom the group consisting of: pancreatic islets, hepatic cells, neuralcells, renal cortex cells, vascular endothelial cells, thyroid andparathyroid cells, adrenal cells, thymic cells, ovarian cells andchondrocytes. In some embodiments in which encapsulated cells areproliferating cells, the proliferating cells may be stem cells,progenitor cells, proliferating cells of specific organs, fibroblastsand keratinocytes or cells derived from established cell lines, such asfor example, 293, MDCK and C2C12 cell lines. In some embodiments,encapsulated cells comprise an expression vector that encodes one ormore proteins that are expressed when the cells are maintained. In someembodiments, the protein is a cytokine, a growth factor, insulin or anangiogenesis inhibitor such as angiostatin or endostatin, othertherapeutic proteins or other therapeutic molecules such as drugs.Proteins with a lower MW, less than about 60-70 kD, are particularlygood candidates because of the porosity of the gel-network. In someembodiments, the cells are present as multicellular aggregates ortissue.

Self gelling alginates may be used to produce alginate gels greater than5 mm with a homogenous alginate network. In some embodiments, thehomogenous alginate gel is greater than 10 mm. The gel formed by thediffusion methods are generally not homogenous alginate gels greaterthan 1 mm. In preferred embodiments, the homogenous alginate gel formedby self gelling alginate gel formation is free of sulfates. citrates,phosphates (TSPP: Tetra sodiumPyroPhosphate and Polyphosphate are usedin food applications with alginate puddings etc.), lactatates, EDTA(Ethylenediaminetetraacetic acid) and lipids as with liposomes used toencapsulate gelling ion.

There are numerous applications for self-gelling alginate. In someembodiments, the self-gelling alginate is used in food products. Theself-gelling alginates that are particularly useful in those foodproducts which are prepared as a liquid/slurry mixture with other foodingredients and is dispensed into a vessel. The vessel is preferably amold where the gel/food product sets to form a solid or semi-solid witha molded shape. Candies, edible decorations, puddings and other moldedshape food products can be prepared.

In some embodiments, the self-gelling alginates are used in biomedicalapplications. Biocompatible self-gelling alginates may be appliedtopically. The biocompatible self-gelling alginates are particularlyuseful in those biomedical applications in which it is desired for thegel matrix to conform to a space in situ such that the self-gellingalginate can be dispensed as a dispersion into the site where the matrixis desired. The dispersion fills the cavity or space in liquid/slurryform and sets to form a solid within the cavity or space. Alternatively,the dispersion be dispensed topically where it can be spread prior tosetting. In some embodiments, the self-gelling alginates are used in themanufacture of matrices which can be prepared with specific shapes bypreparing a liquid/slurry mixture that is dispensed into a mold where itsets to form a solid with a molded shape and/or to prepare matrices withencapsulated cells useful as tissue or organ replacements.

Alginate self-gelling systems that are controllable, biocompatible andparticularly designed for in situ gel formation implantation purposesare provided. Solutions that can easily be used for injections orapplied in other ways inside or outside the body are provided which setto form solid gel matrices. By mixing an alginate in the presence of asolvent with a gelling ion source of which the gelling ions are boundwithin the gel network of an insoluble particle, the gel formingmaterial can be dispensed as a liquid and set in a desirable pattern andtime frame. The solution at a predefined time hardens and forms a gel.The formulation is biocompatible, as variations in pH and presence oftoxic compounds are omitted. Significant deviations from biologic pH areunnecessary.

In some embodiments, the self gelling alginate is used in biomedicalapplications such as tissue bulking such as for the treatment of refluxproblems (i.e. treatment of incontinence, renal reflux or esophagealreflux problems), embolization such as in the treatment of benign ormalignant tumors, anti-adhesion treatment as post-surgical procedures,and wound treatment. The current technology may be used in severalapplications, including tissue constructs ex vivo or in vivo, as cellsor other biomaterials may be mixed into the gelling system therebycreating a bioartificial extracellular matrix supporting cells ortissue. According to some applications, biocompatible solid depots maybe implanted which release active ingredients such as proteins and drugsover time.

The self gelling alginate is particularly useful as a tissue bulkingmaterial in that it can be introduced to a site that is remotelyaccessible and dispensed as a liquid slurry to more fully conform to acavity relative to other types of implants. The dispersion can bedispensed in an amount sufficient to displace and support other tissuesor organs in the body and upon formation of a gel in situ providestructure to maintain and support the other tissue or organs. The selfgelling alginate may comprise components that make it well suited fortissue bulking applications. For example, the use of strontium as agelling agent will result in a gel that inhibits cell overgrowth andunwanted tissue formation.

The self gelling alginate is particularly useful in embolizationprocedures in that it can be introduced to a blood vessel that isremotely accessible and dispensed as a liquid slurry to fully conform tointerior or the blood vessel and more fully and effectively block it offrelative to other types of closures such as sutures. The dispersion canbe dispensed in an amount sufficient to block off circulation uponformation of a gel in situ. The self gelling alginate may comprisecomponents that make it well suited for embolization applications. Forexample, the components can be selected for relatively fast setting andhigh strength. The self gelling alginate used in embolizationapplications may include contrast reagents to monitor its presence andlocation.

The self gelling alginate is particularly useful in anti-adhesiontreatment as post-surgical procedures in that it can be introducedthroughout the area of surgical intervention as a liquid slurry to fullycover exposed surfaces particularly at or near incision sites. The selfgelling alginate may comprise components that make it well suited foranti-adhesion applications. For example, the use of strontium as agelling agent will result in a gel that inhibits cell overgrowth andunwanted tissue formation.

The self gelling alginate is particularly useful wound treatment in thatit can be introduced throughout the area of wound as a liquid slurry tofully cover exposed surfaces. In addition, the self gelling alginate canbe dispensed internally through the wound site for example as a liquidslurry. The dispersion can be dispensed in an amount sufficiently tofill the internal cavity whereupon formation of a gel in situ the gelwill block off any internal wounds and prevent blood loss throughinternal bleeding. The self gelling alginate may comprise componentsthat make it well suited for wound treating applications. For example,blood clotting components as well as antiseptic and antibioticcompositions may be included.

The self gelling alginate is particularly useful to produce tissueconstructs ex vivo or in vivo. Cells or other biomaterials may be mixedinto the gelling system thereby creating a bioartificial extracellularmatrix supporting cells or tissue. The dispersion can be introduced insitu as a liquid slurry to a site where the tissue/cells can function toachieve a therapeutic effect. Examples of tissue constructs includebone, cartilage, connective tissue, muscle, liver, cardiac, pancreas andskin. Examples of this may be preparations containing insulin-secretingcells for the treatment of diabetes, formulations containingchondrocytes for the repair of defective joints, and cells for treatingParkinson's disease. Such cells can be incorporated into the liquidslurry and dispensed into the site where upon gel formation they willexist and function within a biocompatible alginate matrix. The gel mayalso be used as an immune-barrier protecting entrapped cells against thehost immune system. Self-gelling alginate may also be used toencapsulate cells ex vivo whereby the gel can be formed into a shapecompatible with its intended use. In some embodiments, the self-gellingalginate may be used to encapsulate cells, such as dermal cells, andprepare artificial skin such as that which is used to treat burn victimsand others in need of skin grafts or large area wound healing. In someembodiments, the self-gelling alginates may be used to encapsulate cellsand form matrices which can be implanted.

The treatment of diabetes may comprise the production of a biocompatiblematrix comprising insulin producing cells by preparing dispersioncomprising insoluble alginate/gelling ion particles and insulinproducing cells in a solution of soluble alginate and dispensing thedispersion to a site in an individual's body where the biocompatiblematrix forms. The site within the individual body may be a cavity or astructure implanted within the individual. The dispersion may bedispensed into a mold, structure or container where is forms abiocompatible matrix which is implanted into the body of an individual.The insulin produced by the cells in the matrix is secreted by the cellsand released from the matrix into the body of the individual where itfunctions to alleviate the symptoms of the diabetic condition. In someembodiments, the insulin producing cells are pancreatic islet cells. Insome embodiments, the insulin producing cells are recombinant cellsproduced to express and secrete insulin.

The self gelling alginate is particularly useful to produce coateddevices such as implantable devises. In some embodiments, the device isselected from the group consisting of: a stent, a cardiac pacemaker, acatheter, an implantable prosthetic, a surgical screw, a surgical wire,a tissue bulking implant, an esophagus reflux inhibiting implant, anincontinence inhibiting implant, a renal reflux, a container suitablefor holding cells that are deposited on the exterior of a surface and/orencapsulated with an alginate matrix such as a solid device ormacrocapsule, a breast implant, a chin implant, a cheek implant, apectoral implant, a gluteus implant and a dental implant. The coatingusing self gelling alginate produces an effective coating regardless ofshape. The use of strontium as gelling ion is particularly useful toinhibit cell overgrowth upon implantation.

Self-gelling alginates may be used in the manufacture of matrices whichcan be implanted. Such matrices can be prepared with specific shapes bypreparing a liquid/slurry mixture that is dispensed into a mold where itsets to form a solid with a molded shape. Matrices prepared forimplantation may comprise biologically active agents and/or cell. Thegels may be produced and implanted surgically, applied topically or intoan organ through external openings.

According to some embodiments, kits are provided for producing analginate gel. The kits may comprise a first container comprising solublealginate; and a second container comprising insoluble alginate/gellingion particles. The individual containers may be separate containercompartments of an integrated container system.

In some embodiments, the kits comprise soluble alginate in the form of asolution. In some embodiments, the kits comprise soluble alginate freeof a solvent. In some embodiments, the kits comprise an additionalcontainer comprising a solvent.

In some embodiments, the kits comprise insoluble alginate/gelling ionparrticles in the form of a powder. In some embodiments, the kitscomprise insoluble alginate/gelling ion parrticles in the form of adispersion.

In some embodiments, the kits comprise an additional containercomprising a drug, a peptide, a protein, a cell, a detectable label or acontrast reagent. In some embodiments, the kits comprise a drug, apeptide, a protein, a cell, a detectable label or a contrast reagentincluded in the container comprising soluble alginate solution or powderand/or in the container comprising insoluble alginate/gelling ion powderor dispersion.

According to some embodiments, compositions are provided for preparing agel. The composition comprises an immediately soluble alginate andinsoluble alginate/gelling ion particles. The composition may furthercomprise a drug, a peptide, a protein, a detectable label or a contrastreagent. The composition may be a component in a kit. Such a kit mayfurther comprise a container with a solvent.

Kits preferably contains instructions for use.

In some embodiments, the kits comprise a mixing device. Mixing devicesmay be integrated as part of a container or container system. In someembodiments, the mixing device comprises a valve system which allows forpassage of the dispersion from one container to a different container tofacilitate mixing.

In some embodiments, the kits comprise a dispensing device. Thedispensing device may be an applicator in communication with a mixingdevice and/or a container adapted for containing the dispersion. In someembodiments, the dispensing device comprises a catheter. In someembodiments, the dispensing device comprises a syringe.

EXAMPLES Example 1 Gelling with Different Calcium Concentrations

In this experiment gels were made by mixing a solution of sodiumalginate (Protanal SF 120) and a calcium alginate dispersion (ProtaweldTX 120). The amount of calcium alginate was varied (1.0%, 1.5% or 2.0%in the gel), while the amount of sodium alginate was constant (1.0% inthe gel). The setting of the self gelling system during time wasmeasured by using a Physica MCR 300 rheometer (Measuring system: PP50,serrated, Temperature: 20° C., Gap: 1 mm, Frequency: 1 Hz, Strain:0.005). The solution and dispersion were mixed immediately beforeaddition of a 3 ml sample to the rheometer, and the oscillation test wasperformed over a period of 18-24 hours.

As shown in FIG. 1, the gel strength increased rapidly with time duringthe first 1-2 hours, and thereafter the change in gel strength wasreduced as the gel showed a tendency to stabilize. The data also showsthat the gel strength was increased at the higher calcium concentration.

Example 2 Gelling with Different Alginate Concentrations

Alginate self gelling systems were made by mixing a solution of sodiumalginate (Protanal SF 120) with a suspension of calcium alginate andmeasurements performed as described in Example 1. Oscillationmeasurements were made over a period of 18-24 hours. Equal amounts ofsodium alginate (Protanal SF 120) and calcium alginate (Protaweld TX120) were used. The amount of sodium alginate and calcium alginate waseach adjusted to be 0.75%, 1%, 1.25% and 1.5% in the final gelrespectively (FIG. 2). The gelling kinetics followed a similar patternin all four cases. However, as in Example 1 the gel strength clearlyincreased with increasing alginate concentrations.

Example 3 Gelling with Alginates of Different Molecular Weight

In this experiment the gelling kinetics for sodium- and calciumalginates with different molecular weight was compared (FIG. 3). Areduced MW sample of the alginate (Protaweld TX 120) was obtained byincreasing the temperature for several days (FIG. 3, Panel A) ProtanalSF 120 and Protanal SF/LF sodium alginates with different MW were alsocompared (FIG. 3, Panel B). Rheological measurements were performed asdescribed in Example 1 and the data are shown in FIG. 3. As shown in thefigure the gelling process was clearly dependent upon the alginatemolecular weight both for sodium and calcium alginate. In both cases thegel strength increased more rapidly for the high molecular weightalginate and also reached a higher level. Similarly to what is seen inFIG. 3, FIG. 11 also shows gelling of a reduced MW sample of sodiumalginate (PRONOVA UP G 100) obtained by increasing the temperature.However, in this case gelling was initiated by mixing with strontiumalginate. As shown in FIG. 11 the gelling process was clearly dependentupon the alginate molecular weight as the gel strength reached a higherlevel for the high molecular weight alginate. The data FIG. 11, whichused calcium alginate or striontium alginate at 100% saturationstoichiometry, also shows that increasing the alginate concentration maycompensate for the reduction in MW with regards to gel strength.

Example 4 Gelling with Different Gelling Ions

In this experiment calcium or strontium alginate was mixed with sodiumalginate (Protanal SF 120). Calcium and strontium alginates were made bykneading alginic acid with calcium carbonate. Rheological measurementswere performed as described in Example 1 and the data are shown in FIG.4. The amount of sodium alginate and strontium/calcium alginate was eachadjusted to be 0.75% in the gel. Clearly, the use of strontium asgelling ion gave rise to a stronger gel structure as well as a fastergelling kinetics.

Example 5 Gelling with Different Content of Guluronic Acid

As the content of guluronic acid in alginates are known to have majorinfluence on the gel strength of alginate gels the effect of usingsodium alginates (Protanal SF 120 and Protanal HF 60D) with differentcontent of guluronic acid was tested. In FIG. 5, panel A is shown thestorage modulus as a function of time for gels containing sodiumalginate with a high or low content of guluronic acid. In both curvesthe system was gelled by mixing with a dispersion of calcium alginate(Protaweld TX 120) with a high content of guluronic acid. The amountcalcium alginate and sodium alginate used was adjusted to be 1.0% ofeach in the gel. Measurements were performed as described in Example 1.Clearly the use of a sodium alginate with a high content of gluronicacid increased the gel strength of the system although in both cases acalcium alginate with a high content of guluronic acid was used. In FIG.5, panel B, strontium alginate (FMC process product with a high contentof guluronic acid per Example 14) was also mixed with sodium alginateswith a high and low content of guluronic acid. The sodium alginates usedwere PRONOVA UP 100G (69% G, MW: 122 000) and PRONOVA UP 100M (46% G,MW: 119 000). The MW (and viscosity) of the two sodium alginate batcheswas selected to be similar (as close as possible). As the data clearlyshows, also when using strontium alginate as the gelling ion source, theuse of sodium alginate with a high content of guluronic acid increasedthe gel strength compared to a sodium alginate with a low content ofguluronic acid.

Example 6 Stability of Gels Made with Different Calcium Content UnderPhysiologic Conditions

In this example, stability and biodegradability of alginate gels madewith different content of calcium ions was observed (FIG. 6). Gels discswere made by mixing calcium alginate (Protaweld TX 120) autoclaved forsterility and a sterile filtered sodium alginate (Pronova UP LVG) to afinal concentration of 1.0% and 0.7% respectively. The dispersions wasgelled into two Petri dishes. The gel discs in one dish (marked V) wasafter initial gelling washed with 50 mM calcium chloride for 10 minutesand both dishes was thereafter added cell culture medium (DMEMsupplemented with 10% FBS). The medium in the dishes was then changedwith fresh medium regularly three times a week and the dishes werestored in a CO₂ incubator at 37° C. under sterile conditions. Theinitial size of the largest gel disc in each dish was of the same size.After six months a major fraction of the gel not washed with calcium asshown in FIG. 6 disappeared while the gel discs washed with additionalcalcium remained with little or no change in size during time. Thisclearly shows that the alginate gel made with a limited content ofcalcium may be strongly degraded under physiologic conditions.

Example 7 Cell Entrapment

Encapsulation of cells in alginate microbeads is a widely used techniquecurrently under development for different biomedical applications.Alginate beads are used as a “biofactory” for therapeutic substances.The alginate gel allows the influx of essential nutrients like oxygenand glucose and the efflux of desired therapeutic molecules and wasteproducts. By using responsive cells like islet cells the “biofactory”responds to the host. However, the gel network needs to protect theentrapped cells from the immune system of the host which is highlycritical when implanting foreign cells into the body. It has, however,been shown that cells may be successfully entrapped in other alginatestructures than microbeads.

The effect of the gel on cells entrapped in our self gelling alginatematrix are also shown (FIG. 7). In one of the experiments (FIG. 7, panelA) C2C12 mouse myoblast cells were mixed with PRONOVA UP MVG alginatesolution before mixing with an autoclaved calcium alginate dispersion(Protaweld TX 120). The mixture containing cells and 0.7% sodiumalginate and 1.0% calcium alginate was injected into a Petri-dish andmolded as disks. After a few minutes the gel was washed with 50 mMcalcium chloride for 10 minutes in order to prevent degradation of thegel (see Example 6) and the cell growth medium (DMEM supplemented with10% FBS) was thereafter added to the gel. The alginate gel/cell culturewas stored in a CO₂-incubator at 37° C. under sterile conditions and themedium was thereafter regularly changed three times a week. After 45days in culture the presence of viable cells were visualized undermicroscope by calcein staining. A fluorescence microscope was used tovisualize living cells.

The picture shown in FIG. 7, panel A, shows the presence of viable cellsboth outside and inside the gel. Because of different focusing in themicroscope on different part of the gel different spots are more or lessclear in the picture. Numerous viable cells or small cell colonies canbe seen inside the gel as small enlightened spots. The large enlightenedarea covering a large part of the picture shows viable cells that hasentered the gel surface and multiplied there. This part of gel surfaceis covered completely with cells growing as monolayers. Some cellaggregates on the gel surface are, however, also present in other areas.

In another experiment (FIG. 7, panel B) human chondrocytes wereentrapped in alginate self-gel. In this case the gel was made of 5 mlmixed self-gel of PRONOVA SLG 20 (low viscosity lyophilized sterilealginates with high guluronic acid content) and calcium alginate (FMCprocess product, Example 14) containing human chondrocytes. Three daysafter gelling the gel was sectioned into 600 μm slices using avibratome. The gel slices was stored in cell growth medium in aCO₂-incubator and the picture was taken after six months. The picturewas taken using a fluorescence microscope after staining the cells withcalcein as an indicator of cell viability. The picture clearlydemonstrates the presence of a high number of viable cells. The alginategel network therefore must be a good matrix in supporting the cells fora long period of time. In conclusion the data clearly demonstrates thatthe gel may be a biocompatible matrix for cells and cell growth.

A series of different alginate samples containing human chondrocyteswere also prepared. In this case higher molecular weight alginates wereselected in order to retain gel strength at low gelling ion and alginateconcentrations. These were PRONOVA SLG 100 and PRONOVA SLM 100 (highviscosity lyophilized sterile alginates with high and low guluronic acidcontent respectively). Chondrocytes were mixed into an approximately 2%solution of alginates in cell medium. The alginate/cell suspensions werethen maintained for about half an hour in order to allow release of airbubbles before further use. The cells suspensions were then mixed withinsoluble calcium or strontium sterile alginates (FMC process productprepared per Example 14, in vials containing 5 ml 10% alginatedispersion, totally 0.5 mg alginate). Each vial of insoluble alginatecontained a magnet for stirring and was used on the same day afteropening. The insoluble products had also been milled and sifted in orderto control the particle size and were manufactured as strontium andcalcium alginates with a high or low guluronic acid content. The mixingof the alginate/cell suspension and insoluble alginate dispersion weredone in small volumes in small test tubes. The insoluble alginatedispersions were kept under magnet stirring when the desired volumeswere taken out of the vials. Different samples were mixed as describedin the table below. Gel systems containing cells (start concentrationsin parenthesis) Group Alginate solution Ions source alginates MixingAlt. 1 1.6% PRONOVA 2.0% Strontium high G (10%) 4:1 SLG 100 (2.0%) Alt.2 1.6% PRONOVA 2.0% Calcium high G (10%) 4:1 SLG 100 (2.0%) Alt. 3 1.6%PRONOVA 2.0% Strontium high G (10%) 4:1 SLM 100 (2.0%) Alt. 4 1.6%PRONOVA 2.0% Strontium high M (10%) 4:1 SLG 100 (2.0%)After an initial gelling of the mixture for a few minutes the small gelpieces were stored in the cell culture medium in a CO₂ incubator andcell viability was checked with calcein staining (as previouslydescribed) after a week. In this study, good cell viability in all thealginate gel/cell samples was observed. Alternatively,alginate/chondrocyte samples can be prepared in situ. Depending on theapplication, different self-gel formulations may be adapted for eachparticular use. The gel may contain cells directly but may also containmicrobeads or other biostructures containing cells. The formulation maybe injected before the gelling has completed but the gel could also beallowed to set ex vivo, either completely or partially beforeimplantation. Furthermore, the gel may be made more or less strong orporous in order to allow cell proliferation within the gel or not, toadapt to the environment or give immunoprotection. Depending of the typeof gelling ion and type of alginate the gel can be formulated to be lessattractive for the overgrowth of cells. Also the gel structure may bemade more or less biodegradable by using a low calcium content (as shownin Example 6 and FIG. 6), low molecular weight alginate or low alginateconcentrations. The gel may also be mixed with other biopolymers likehyaluronate or chitosan for improved properties. The gel may also befurther strengthened by applying additional calcium to the constructthrough a suitable soaking or spraying procedure.

Example 8 Controlled Release Systems

The usefulness of alginate in controlled release systems for thedelivery of drugs or other therapeutic molecules has been demonstrated.The type of gel preparations demonstrated here may also be usedsimilarly and have advantages in different formulations. One example isthe use of biodegradable gels, i.e. by using a low concentration ofgelling ions in order to limit the treatment period. In the treatment ofcancer patients a space-filling gel containing drugs or radioactiveisotopes may be applied during surgical procedures in order to preventrecidive of the disease. After the active substances are released orradioactivity has decayed it may be desirable that the gel dissolves andis excreted from the body. Self-gelling alginate controlled deliveryformulations may of course also be injected directly into the bodywithout any surgical procedures and the gel/alginate solution may alsobe used for oral drug delivery. For oral use alginate is currently wellknown in formulations as an anti-reflux remedy. It is therefore alsopossible that alginate self-gelling formulations may find similar uses.

Example 9 Tissue Engineering Applications

The entrapment of cells within the alginate gel as presented here may beused to produce implantable “biofactories” excreting active substancesfor the treatment of a variety of diseases. However, the entrapment ofcells within the alginate gel may also be used in tissue engineeringapplications. For tissue engineering the growth of cells within or on3-dimensional constructs is needed and therefore good biomaterials forsuch applications are needed. The time-delayed release of cross-linkingions allow the gelling-ion alginate suspension to be molded into complexgeometries before gelation occurs. Under ex vivo conditions suchalginate structures may be used as a growth substrate in the developmentof tissue or artificial organs. Cells grow on the surface of alginategel beads as the gel surface may be a growth substrate for cells. Thegrowth of cells on alginate gels have been found to be dependent of thealginate and the gelling ions used. The present self-gelling formulationmay be used to create multiple layers of cells growing inside or on thesurface of alginate sheets or other shaped gel structures. Furthermore,the alginate gel may later be removed through treatment with citrate,phosphates or other gelling ion chelating agents. This gives thepossibility to combine several cell layers in the construction oftissues or organs. Several types of cells inside or on the surface ofgel structures may be combined if this is desirable for the developmentof the construct.

Nerve regeneration is an interesting example of the use of alginatewithin tissue engineering. The filling of artificial nerve conduits withself-gelling alginate may be suitable for the creation of constructswith improved guidance and biocompatibility for nerve regrowth. Thissystem may give better flexibility and better control over moldingprocesses and structure properties as compared to other techniques.

Injectable alginate/cell suspension systems may also be delivered to thedefective or damaged tissue site even without surgical intervention. Forsuch applications it may be critical to have a certain working time toshape the material before it gels. However, the gelation rate may alsobe required to be reasonable rapid so that a prolonged patient waitingtime or problems with applying the gel/solution can be avoided. Theself-gelling system may as shown here and previously mentioned beadapted with different gelling time-curves and different strength andstability properties. This variability may therefore be used to adapt toeach type of injection procedures. As an example the repair of cartilagedefects holds a potential for the use self-gelling alginate structures.Alginate has been found to be a useful biomaterial to be used forcartilage tissue engineering, and it has been found that alginate maystimulate chondrogenesis. Therefore self-gelling alginate solutions withor without chondrocytes or other cells may be directly injected in thetreatment of articular defects. Osteoarthritis patients are alreadytoday being treated with “joint fluid therapy” and there are twoproducts on the market, sodium hyaluronate (Hyalgan) and hylan G-F 20(Synvisc) which are believed to work as lubricants by supplementinghyaluronic acid, the substance that gives joint fluid its viscosity.Pain relief lasts as long as six to 13 months in some people. Thetherapies have proven most effective for people with mild to moderateknee osteoarthritis. However, as hyaluronic acid is known to be degradedin the body the use of other biopolymers like alginate with lessbiodegradability and good biocompatibility provide advantages.

Alginate hydrogels may be lyophilized or the water be removed partly orfully in other ways treated in order to create biocompatible structureslike sponges or fibers. The use of the technology presented here, usingself-gelling alginate systems, may also be used as a step in themanufacturing of biocompatible sponges or other structures which areuseful for tissue engineering or other applications.

Self gelling alginate formulations may be used in the coating of stentsor grafts or other implantation devices. Depending of the type ofalginate formulation the coating layer may be made more or lessbiodegradable and give more or less support for the ingrowth of hostcells or the growth of cells added to the device.

Example 10 Tissue Bulking

Alginate may be delivered into the submucosa proximal to the urethralsphincter to provide bulking for the treatment of bladder incontinenceand procedures has already been performed in the clinic. Another examplemay be the delivery of alginate formulations at the junction between theesophagus and the stomach to aid in the treatment of gastro-esophagealreflux disorders. The high degree of compatibility of alginates makesthe use as an injectable solution in cosmetic procedures an attractivealternative to other materials.

Formulations based upon self-gelling alginate systems may be used tocreate injectable solutions or pastes with predefined hardening timewith purpose of filling a predefined volume. As previously mentioned gelformulations may be made more or less biodegradable giving the bulkingformulation a desirable property.

Example 11 Embolization of Blood Vessels

Methods for forming endovascular occlusions may be used to treatconditions such as arteriovenous malformations, aneurysms, excessiveblood supplied to tumors, control of massive vascular hemorrhaging, andother conditions which require an embolization to alleviate thecondition. Some embolic systems include the use of polymers solutionswhich begin to solidify or precipitate when contacted with blood orother bodily fluids. Such systems, however, suffer from the problem ofthe polymer solution migrating into undesired parts of the body becauseof the time delay necessary to cause formation or precipitation of thesolid polymer. Migration in these polymer solution systems isparticularly problematic when the solution is injected into “high flow”areas, such as vascular systems. Fibers formed from polymer solutionsystems also tend to suffer from other problems, such as not embolizingwell, being overly brittle, or not being biocompatible. The use ofparticles or beads of PVA (Poly vinyl alcohol) or gelatin beads havebeen found useful for embolization and are currently used in clinics.

Alginate based formulations have also been proposed for use inembolization procedures. It has been suggested that endovascularocclusions may be induced using calcium alginate by controlling theinjections of an alginate liquid and a calcium chloride solution to meetand polymerize at a site within the vascular system targeted forocclusion. Compared to such systems, the use of self-gelling alginateformulations as presented here have advantages. Treatment may beperformed as single injections and the strength of the self-hardeningformulation may be adjusted with better control of the system. Inparticular self-gelling alginate formulations are useful when the timebefore gelling and biodegradability needs to be controlled.

Example 12 Anti-Adhesion Formulations

Formation of adhesions are attributable to surgical operations, trauma,infections etc. Adhesions frequently occur after abdominal operationsand represent a major clinical problem resulting in intestinalobstruction, infertility, and pain. Efforts to prevent or reduceadhesions have largely been unsuccessful; however, recently developedmechanical barriers using different biopolymers have demonstratedclinical progress in adhesion prevention.

Alginate based formulations have also been proposed as anti adhesionbarriers. Anti-adhesion barriers may be formulated by using theself-gelling alginate system presented here. The solution/gelformulation is premixed immediately before use and made with suitablebiodegradability. Such types of formulations may also include otherpolymers, drugs or other supportive compounds. Additional polymers maybe used to improve the properties of the gel, among others to increasethe adhesion between gel structure and tissue.

Example 13 Wound Healing Formulations

Alginate dressings are commonly used to treat exuding wounds. Currentalginate products for wound healing are composed of soft, non-wovenfibers or pads. Alginates can absorb many times their own weight andform a gel within the wound to fill in dead space and maintain a moistenvironment. It has also been suggested that alginates may influence thewound healing process through more unknown mechanisms, and it has beenpostulated that calcium present within alginate wound dressing mayinfluence the would healing process through influence on certain cells.

Self-gelling alginate structures are capable of conforming to the threedimensional structure of a tissue surface during healing processes.Among other formulations with a more controllable and defined calciumcontent may be achievable as well as structures with high degree ofresorbability.

Example 14 Insoluble Calcium Alginate Production

A calcium alginate was prepared using an ultrapure (reduced endotoxincontent) commercial alginate. In addition, the calcium content was of astoichiometric nature. Specifically 60 g of PRONOVA UP LVG sodiumalginate (batch FP-008-04) having a molecular weight of approximately130,000, a viscosity of approximately 150 mPas (1% solution, 20° C.), aguluronate content of 64%, and an endotoxin content of 260 EU/gram, wasdissolved in 5 liters of purified water. 26 grams of sodium carbonatewas added. 165 grams of calcium chloride dehydrate was first dissolvedin 500 ml of purified water and the pH was adjusted with nitric acid toneutrality. The alginate solution was added carefully to the calciumchloride solution under continuous stirring. The precipitated calciumalginate was then washed successively 4 to 8 times with purified wateruntil the conductivity was reduced to a level similar to that ofpurified water. The washed calcium alginate was then dried under vacuumand subsequently milled. Insoluble strontium alginate may be prepared bysimilar a method using strontium salt in place of calcium chloride.Resulting insoluble alginates have controlled stoichiometric or substoichiometric amounts of calcium or strontium which when used in thegelling systems produce gels of reproducible consistency greater thanthose made using insoluble alginates produced by other methods.

Example 15 Gelling in the Presence of Other Ions and Calcium BindingAgents

In the experiments we performed oscillation measurements as describedearlier (Example 1). Storage modulus was measured as a function of timefor gels containing 1.25% sodium alginate (PRONOVA UP 100 G) mixed with5.5% strontium alginate (Example 14) at a ratio of 4:1 (final alginateconcentration was 2.1%). The development of the gel was measured in thepresence or absence of sodium chloride or sodium hexametaphosphate (FIG.8). Two different concentrations of sodium chloride was tested and thedata clearly demonstrates an increased gelling rate when increasing theconcentration of sodium ions. The presence of a calcium binding agentlike sodium hexametaphosphate clearly also changed gelling kinetics andreduced the final strength of the gel. The data thus show that thepresence of non gelling ions like sodium or calcium complexing compoundslike hexametaphosphate may be used to modify gelling kinetics and finalproperties of the gel.

Example 16 Gelling with Different Sized Calcium Alginate Particles

During the manufacturing process for insoluble alginates the particlesize of the final product may be controlled. In this example we made onebatch of calcium alginate that was milled and sifted through differentfilters in order to separate between different particle sizes. When thedifferent strontium alginate particles were gelled with sodium alginateunder otherwise identical conditions there was a large difference in thegelling process and final properties of the gel (FIG. 9). While thegelling was very rapid for the smaller particle sizes, the total gellingtime was considerably longer for the larger particle sizes and this alsogave gels with much higher strength. However, for the smaller particlesizes it should be noted that some degradation of the gel probablyoccurred during mixing of the two components as the gelling speed wasvery rapid (in particular for particles less than 25 μm). This effectmay therefore also contribute somewhat to the difference in final gelstrength. Nevertheless, in conclusion our data shows that the particlessize needs to be accounted for and may be actively used in order toobtain desirable properties.

Example 17 Gelling at Different Temperatures

We also tested whether adjusting the temperature could be actively usedto control the setting of the gel system. In FIG. 10 is shownrheological data for a mixture of calcium alginate and sodium alginateat different temperatures. Clearly, the gelling rate was reduced at 10°C. and also increased at 37° C. as compared to room temperature. Thedata thus shows that the temperature may be actively used in order tocontrol gelling kinetics. Among others this could be actively used byreducing the temperature to allow more time for gel preparation andhandling before administration in vivo.

Example 18 Gelling with Non-Saturated Calcium or Strontium AlginateParticles

Calcium alginates and strontium alginates were prepared which werestoichiometric non-saturated with gelling ions (less than 100%saturated). Such particles in contrast to the saturated particlesrehydrated very rapidly in contact with water containing solutions. Thenon-saturated particles therefore could be used to form an instant gelstructure consisting of gel particles (non solid gel). Although the gelstructure was weak it was easily shapeable for a long period. Cells orother materials could easily be mixed into the gel structure by simplyadding them to the water containing solution before mixing in thepowder. Also other particles or materials could be mixed into the gelstructure by premixing with the non-saturated particles before addingthe water containing solution. One example of this is to premix drysodium alginate, saturated insoluble alginate and non-saturatedinsoluble alginate before the water containing solution is added. Incontact with water this mixture as previously will give an instant waterabsorbing gel structure, however, the gel strength will also increasefurther with time as the particles of soluble alginate and insolublesaturated alginate in the gel gradually starts to hydrate and gel upondissolving. The formulations mentioned here, utilizing the waterabsorbing properties of non-saturated insoluble alginates may thus alsobe used in combinations with cells or other materials and be very usefulfor tissue engineering and other applications.

Example 19 FP-411-03, Production of Calcium Alginate with StoichiometricAmount of Calcium (100% Saturation), G-Rich Alginate, with SodiumCarbonate and Nitric Acid

50 grams of PRONOVA UP LVG sodium alginate, batch FP-008-04 (64%guluronic acid content, 130,000 g/mol molecular weight, 146 mPasviscosity of a 1% solution at 20° C., 400 EU/gram endotoxin content)were dissolved in 3 liters of purified water. 15 grams of sodiumcarbonate was added. A calcium solution was added consisting of 139grams of CaCl₂.2H₂O dissolved in 300 ml of purified water with theaddition of 12 ml HNO₃ (65%). A fine precipitation resulted. Theconductivity of this solution was measured to be 55 mS/cm. Theprecipitate was washed successively (8 times) with purified water untilthe conductivity was 0.08 mS/cm. The precipitate was vacuum dried.

Example 20 FP-411-04, Production of Calcium Alginate withSub-Stoichiometric Amount of Calcium (50% Saturation), G-Rich Alginate,with Sodium Carbonate and Nitric Acid

25 grams of PRONOVA UP LVG (description as given in Example 19) wasdissolved in 1.5 liters of purified water. To calculate the amount ofcalcium required, 25 grams of alginate represent 0.146 mol of alginate(using a monomer molecular weight of 171 g/mol). The alginate used,PRONOVA UP LVG batch FP-008-04 has a guluronic acid content of 64% whichresults in 0.093 mol of calcium-binding sites (0.146 mol of alginate×64%guluronic acid monomers). For a 50% substitution with calcium this wouldrequire 0.0465 mol of calcium (0.093/2=0.0465 mol). 0.0465 mol ofcalcium salt is calculated to be 6.84 grams of the dihydrate (0.0465mol×147.02 g/mol (CaCl₂.2 H₂O)=6.84 g of CaCl₂.2H₂O. 6.84 grams ofCaCl₂.2H₂O are dissolved in 150 ml purified water and 6 ml of HNO₃(65%). 7.5 grams of sodium carbonate are added to the alginate solution.A fine precipitation results. The conductivity is measured to be 8mS/cm. The precipitate is washed successively (6 times) until theconductivity is 0.4 mS/cm. The precipitate is vacuum dried.

Example 21 FP-411-05, Production of Strontium Alginate withStoichiometric Amount of Strontium (100% Saturation), G-Rich Alginate,with Sodium Carbonate and Nitric Acid

47 grams of PRONOVA UP LVG sodium alginate, batch FP-008-04 (64%guluronic acid content, 130,000 g/mol molecular weight, 146 mPasviscosity of a 1% solution at 20° C., 400 EU/gram endotoxin content)were dissolved in 3 liters of purified water. 15 grams of sodiumcarbonate was added. A strontium solution was added consisting of 252grams of SrCl₂.6H₂O dissolved in 300 ml of purified water with theaddition of 12 ml HNO₃ (65%). A fine precipitation resulted. Theconductivity of this solution was measured to be 78 mS/cm. Theprecipitate was washed successively (8 washes) with purified water untilthe conductivity was 0.0159 mS/cm. The precipitate was vacuum dried.

Example 22 FP-411-06, Production of Strontium Alginate withSub-Stoichiometric Amount of Strontium (50% Saturation), G-RichAlginate, with Sodium Carbonate and Nitric Acid

23.3 grams of PRONOVA UP LVG (description as given in Example 19) wasdissolved in 1.5 liters of purified water. To calculate the amount ofcalcium required, 23.3 grams of alginate represent 0.136 mol of alginate(using a monomer molecular weight of 171 g/mol). The alginate used,PRONOVA UP LVG batch FP-008-04 has a guluronic acid content of 64% whichresults in 0.087 mol of calcium-binding sites (0.136 mol of alginate×64%guluronic acid monomers). For a 50% substitution with strontium thiswould require 0.0435 mol of strontium (0.087/2=0.0435 mol). 0.0435 molof strontium salt is calculated to be 11.6 grams of the hexahydrate(0.0435 mol×266.62 g/mol (SrCl₂.6H₂O)=11.6 g of SrCl₂.6H₂O. 11.6 gramsof SrCl₂.6H₂O are dissolved in 150 ml purified water and 6 ml of HNO₃(65%). 7.5 grams of sodium carbonate are added to the alginate solution.A fine precipitation results. The conductivity is measured to be 22mS/cm. The precipitate is washed successively (3 times) until theconductivity is 0.6 mS/cm. The precipitate is vacuum dried

Example 23 FP-506-03, Production of Strontium Alginate withStoichiometric Amount of Strontium (100% Saturation), M-Rich Alginate,Without Sodium Carbonate and Nitric Acid

50 grams of PRONOVA UP LVM sodium alginate, batch FP-408-01 (44%guluronic acid content, 220,000 g/mol molecular weight, 127 mPasviscosity of a 1% solution at 20° C., <25 EU/gram endotoxin content)were dissolved in 3 liters of purified water. A strontium solution wasadded consisting of 252 grams of SrCl₂.6H₂O dissolved in 400 ml ofpurified. A fine precipitation resulted. The conductivity of thissolution was measured to be 43 mS/cm. The precipitate was washedsuccessively (8 washes) with purified water until the conductivity was0.143 mS/cm. The precipitate was vacuum dried, milled and fractionated.

Example 24 FP-505-05, Production of Strontium Alginate withStoichiometric Amount of Strontium (100% Saturation), G-Rich Alginate,without Sodium Carbonate or Nitric Acid

50 grams of PRONOVA UP LVG sodium alginate, batch FP-408-02 (69%guluronic acid content, 219,000 g/mol molecular weight, 138 mPasviscosity of a 1% solution at 20° C., <25 EU/gram endotoxin content)were dissolved in 3 liters of purified water. A strontium solution wasadded consisting of 252 grams of SrCl₂.6H₂O dissolved in 400 ml ofpurified. A fine precipitation resulted. The conductivity of thissolution was measured to be 40 mS/cm. The precipitate was washedsuccessively (7 washes) with purified water until the conductivity was0.1 mS/cm. The precipitate was vacuum dried, milled and fractionated.

Example 25 FP-505-02, Production of Calcium Alginate with StoichiometricAmount of Calcium (100% Saturation), M-Rich Alginate, without SodiumCarbonate and Nitric Acid

50 grams of PRONOVA UP LVM sodium alginate, batch FP-408-01 (44%guluronic acid content, 220,000 g/mol molecular weight, 127 mPasviscosity of a 1% solution at 20° C., <25 EU/gram endotoxin content)were dissolved in 3 liters of purified water. A calcium solution wasadded consisting of 137 grams of CaCl₂.2H₂O dissolved in 300 ml ofpurified water. A fine precipitation resulted. The conductivity of thissolution was measured to be 45 mS/cm. The precipitate was washedsuccessively (8 washes) with purified water until the conductivity was0.0129 mS/cm. The precipitate was vacuum dried, milled and fractionated.

Example 26 FP-504-02, Production of Calcium Alginate with StoichiometricAmount of Calcium (100% Saturation), G-Rich Alginate, without SodiumCarbonate and Nitric Acid

50 grams of PRONOVA UP LVG sodium alginate, batch FP-408-02 (69%guluronic acid content, 219,000 g/mol molecular weight, 138 mPasviscosity of a 1% solution at 20° C., <25 EU/gram endotoxin content)were dissolved in 5 liters of purified water. A calcium solution wasadded consisting of 231 grams of CaCl₂.2H₂O dissolved in 500 ml ofpurified. A fine precipitation resulted. The conductivity of thissolution was measured to be 43 mS/cm. The precipitate was washedsuccessively (8 washes) with purified water until the conductivity was0.0068 mS/cm. The precipitate was vacuum dried, milled and fractionated.

Example 27 Further Examples of Products Produced with VaryingStoichiometries of Calcium and Strontium Content, as Well as Variationsin the Type of Alginate Used (Guluronate-Rich (G) or Mannuronate-Rich(M)) are Shown in the Attached Table

Alginate type Particle size, Batch Salt Viscosity, mPas Saturation % Na% Ca % Sr μm FP-508-03 Ca G 100% 125, 75, 45, 25 113 FP-506-03 Sr M 100%125, 75, 45, 25 127 FP-505-05 Sr G 100% 125, 75, 45, 25 425 FP-505-02 CaM 100% 125, 75, 45, 25 410 FP-504-02 Ca G 100% 993 FP-502-02 Sr M 100%0.12 21.6 75 663 FP-502-01 Ca M 100% 0.16 9.2 75 663 FP-501-06 Sr G 100%0.58 20.5 149 FP-501-05 Ca G 100% 0.44 8.0 149 FP-501-03 Sr M 100% 110FP-501-02 Ca M 100% 0.22 8.6 110 FP-412-01 Sr M  90% 3.1 14.5 110FP-411-06 Sr G  64% 146 FP-411-05 Sr G 100% 0.46 20.0 146 FP-411-04 Ca G64% 3.4 6.4 146 FP-411-03 Ca G 100% 0.16 9.5 146 16.09.2004 Ca G 100%0.21 10.3 190

1. A kit for producing an alginate gel comprising: a first containercomprising soluble alginate; and a second container comprising insolublealginate/gelling ion particles.
 2. The kit to claim 1 further comprisinga mixing device and/or a dispensing device.
 3. The kit to claim 2wherein the mixing device is a T-connector.
 4. The kit of claim 1further comprising an additional container comprising a solvent.
 5. Thekit of claim 1 further comprising a drug, a peptide, a protein, a cell,a multicellular aggregate, a tissue, a detectable label or a contrastreagent.
 6. The kit of claim 1 wherein there is an excess of solublealginate in comparison to insoluble alginate.
 7. The kit of claim 1wherein the concentration of soluble alginate to insoluble alginate isbetween 5:1 and 1:5.
 8. The kit of claim 1 wherein the insolublealginate/gelling ion particle size is selected from the group consistingof: <25 μm, 25-45 μm, 45-75 μm, 75-125 μm and >125 μm.
 9. The kit ofclaim 1 wherein the soluble alginate and/or the insoluble alginatecomprises high G content alginate.
 10. The kit of claim 1 wherein thesoluble alginate and/or the insoluble alginate has a molecular weight ofabout 5-350 kD.
 11. The kit of claim 1 wherein the soluble alginateand/or the insoluble alginate comprises endotoxin levels of <25 EU/gram.12. The kit of claim 1 wherein the soluble alginate and/or the insolublealginate is sterile.
 13. The kit of claim 1 wherein the soluble alginatecomprises one or more of Na-alginate, K-alginate, PEG-alginate orNH₄-alginate.
 14. The kit of claim 1 wherein the insoluble alginatecomprises one or more of Calcium, Strontium, Barium, Copper Manganese,Lead, Cobalt or Nickel.
 15. A composition for preparing a gel comprisingimmediately soluble alginate and insoluble alginate/gelling ionparticles.
 16. The composition of claim 15 further comprising a drug, apeptide, a protein, a detectable label or a contrast reagent.
 17. Thecomposition of claim 15 wherein the soluble alginate and/or theinsoluble alginate comprises high G content alginate.
 18. Thecomposition of claim 15 wherein the soluble alginate and/or theinsoluble alginate has a molecular weight of 5-350 kD.
 19. Thecomposition of claim 15 wherein the soluble alginate and/or theinsoluble alginate comprises endotoxin levels of <25 EU/gram.
 20. Thecomposition of claim 15 wherein the soluble alginate and/or theinsoluble alginate is sterile.
 21. The composition of claim 15 whereinthe soluble alginate comprises one or more of Na-alginate, K-alginate,PEG-alginate or NH4 alginate.
 22. The composition of claim 15 whereinthe insoluble alginate comprises one or more of Calcium, Strontium,Barium, Copper, Manganese, Lead, Cobalt or Nickel.
 23. The compositionof claim 15 wherein there is an excess of soluble alginate in comparisonto insoluble alginate.
 24. The composition of claim 15 wherein theconcentration of soluble alginate to insoluble alginate is between 5:1and 1:5.
 25. The composition of claim 15 wherein the insolublealginate/gelling ion particle size is selected from the group consistingof: <25 μm, 25-45 μm, 45-75 μm, 75-125 μm and >125 μm.
 26. A method fordispensing a self-gelling alginate dispersion comprising: a) forming adispersion by mixing i) a solution comprising a soluble alginate with aninsoluble alginate/gelling ion particles or ii) immediately solublealginate, insoluble alginate/gelling ion particles and a solvent, and b)dispensing the dispersion whereby the dispersion forms an alginate gelmatrix.
 27. A method for dispensing a self-gelling alginate dispersionwithin an individual comprising the method of claim 26 wherein thedispersion is dispended into the individual.
 28. A method of usingalginate gel as tissue bulking material in an individual comprisingdispensing a self gelling alginate dispersion within an individualaccording to claim
 27. 29. The method of claim 28 wherein the alginategel is used as tissue bulking material for treatment of bladderincontinence, wherein said self gelling alginate dispersion is dispensedinto the submucosa proximal to the urethral sphincter of the individualor the alginate gel is used as tissue bulking material for treatment ofgastro-esophageal reflux disorders, wherein said self gelling alginatedispersion is dispensed at the junction between the esophagus andstomach of an individual.
 30. A method of using a self gelling alginatedispersion in a vascular embolization procedure in an individualcomprising dispensing a self gelling alginate dispersion within anindividual according to claim 27, wherein said self gelling alginatedispersion is dispensed into a blood vessel of an individual.
 31. Themethod of claim 30 wherein the vascular embolization procedure isperformed in the treatment of a benign or malignant tumor, wherein saidself gelling alginate dispersion is dispensed into the blood vessel ofan individual that supplies blood to the tumor.
 32. A method of using aself gelling alginate dispersion to prevent post surgical adhesionformation an individual comprising dispensing a self gelling alginatedispersion within an individual according to claim 27, wherein said selfgelling alginate dispersion is dispensed at a surgical site within anindividual.
 33. A method of using a self gelling alginate dispersion intreating a wound in an individual comprising dispensing a self gellingalginate dispersion within an individual according to claim 27, whereinsaid self gelling alginate dispersion is dispensed into the individualat the site of the wound.
 34. A method of using a self gelling alginatedispersion in the treatment of a wound on an individual's skincomprising dispensing a self gelling alginate dispersion according toclaim 26, wherein said self gelling alginate dispersion is dispensedonto the skin of an individual at a wound site on said skin.
 35. Amethod of using an implantable alginate gel comprising forming selfgelling alginate by dispensing a self gelling alginate dispersionaccording to claim 26 and following gel formation implanting theimplantable alginate gel into an individual.
 36. A method of producingan implantable device comprising dispensing a self gelling alginatedispersion according to claim 26 on to a device.
 37. The method of claim26 wherein said dispersion is further comprises one or more componentsselected from the group consisting of: a drug, a peptide, a protein, acell, a multicellular aggregate, a tissue a detectable label, and acontrast reagent.
 38. An alginate gel having a thickness of greater than5 mm and a homogenous alginate matrix network.
 39. The alginate gel ofclaim 38 wherein said gel has a thickness of greater than 10 mm.
 40. Thealginate gel of claim 38 wherein said gel is free of one or more of:sulfates citrates, phosphates, lactatates, EDTA or lipids.
 41. Analginate gel having a thickness of greater than 5 mm and free of one ormore of: sulfates citrates, phosphates, lactatates, EDTA or lipids. 42.The alginate gel of claim 41 wherein said gel has a thickness of greaterthan 10 mm.
 43. An implantable device comprising a homogenous alginategel coating.
 44. The implantable device of claim 43 wherein said deviceis selected from the group consisting of: a cardiac pacemaker, acatheter, an implantable prosthetic, a surgical screw, a surgical wire,a tissue bulking implant, an esophagus reflux inhibiting implant, anincontinence inhibiting implant, a renal reflux, a container suitablefor holding cells that are deposited on the exterior of a surface and/orencapsulated with an alginate matrix such as a solid device ormacrocapsule, a breast implant, a chin implant, a cheek implant, apectoral implant, a gluteus implant and a dental implant.
 45. A methodof filling or repairing osteochondral defects resulting fromosteoarthritis by dispensing a self gelling alginate dispersionaccording to claim 27 wherein said self gelling alginate dispersionincludes chondrocytes
 46. The method of claim 45 wherein thechondrocytes are autologous.
 47. A method of treating diabetes bydispensing a self gelling alginate dispersion according to claim 27wherein said self gelling alginate dispersion includes insulin-producingcells or multicellular aggregates.
 48. The method of claim 47 whereinthe insulin producing cells or multicellular aggregates are pancreaticislets or cultured insulin-producing cell lines.
 49. A method ofimproving the viability of pancreatic islets, or other cellularaggregates or tissue, following isolation and during storage andtransport by incorporating said islets, or cellular aggregates or tissueinto a self gelling alginate dispersion produced according to claim 26.50. A method of preparing insoluble alginate/gelling ion particlescomprising the steps of: dissolving ultrapure sodium alginate in waterand adding a sodium salt; dissolving a gelling ion salt in water andadjusting pH to neutrality; combining the alginate solution with thecalcium chloride solution under continuous stirring; collectingprecipitated insoluble alginate and washing it with water untilconductivity is reduced to a level similar to that of purified water;drying washed insoluble alginate; and forming particles of said dryinsoluble alginate.
 51. The method of claim 50 wherein the gelling ionis calcium or strontium.
 52. The method of claim 50 comprising the stepsof: dissolving ultrapure sodium alginate in water and adding sodiumcarbonate; dissolving calcium chloride dehydrate in water and adjustingpH with nitric acid to neutrality; combining the alginate solution withthe calcium chloride solution under continuous stirring; collectingprecipitated calcium alginate and washing it with water untilconductivity is reduced to a level similar to that of purified water;drying washed calcium alginate; and milling dry calcium alginate toparticles.
 53. Ultrapure insoluble alginate/gelling ion particles madeby the process of claim
 52. 54. Ultrapure insoluble alginate/gelling ionparticles having endotoxin levels of <25 EU/gram.
 55. The ultrapureinsoluble alginate/gelling ion particles of claim 54 wherein alginate issaturated with gelling ion.
 56. The ultrapure insoluble alginate/gellingion particles of claim 54 wherein the gelling ion is calcium orstrontium.